We studied the processing of dengue virus nonstructural protein NS1 by in vitro transcription/translation, and in vivo as expressed by recombinant vaccinia virus. In vitro, the full length NS1-NS2A precursor was made, and was translocated into dog pancreas microsomal membranes and glycosylated, but NS1/NS2A cleavage did not occur. In vivo, brefeldin A blocked secretion of NS1 but did not inhibit NS1/NS2A cleavage. Taken together, these results suggest that NS1/NS2A cleavage occurs in the Golgi, or in a compartment between the ER and the Golgi. We constructed vaccinia recombinant viruses expressing chimeric preM-NS1(3/8)-NS2A proteins, containing only the 3 or 8 C-terminal amino acid residues of NS1. We observed that the chimera with 8 residues of NS1 was cleaved at the NS1-NS2A junction. Thus, the only portion of NS1 required for NS1/NS2A cleavage is the region containing the 8 C-terminal amino acids. Results with analogous NS5-NS1(3/8)-NS2A chimeras showed that these proteins were partially cleaved to about the same extent. These results suggest that the cleavage efficiency and NS1 target size are both reduced when the NS1-NS2A junction is not translocated into the ER.